PROJECT SUMMARY: Biliary atresia (BA) is a cholestatic liver disease of infancy that is the leading cause of pediatric liver transplantation. While evidence supports a role for an aberrant immune response in the pathogenesis of BA, the exact mechanism of disease remains unknown. This gap in scientific knowledge limits identification of immune biomarkers at diagnosis to stratify patient prognosis. While macrophages (M?) have previously been implicated in both human and murine BA, M? are heterogeneous by nature and the pathogenic subsets required for ongoing hepatic injury have not been identified. Overcoming this gap in scientific knowledge will identify the BA-specific pathogenic M? subsets and lead to novel cell-subset specific biomarkers predictive of patient prognosis. We have been the first to identify 3 novel hepatic M? subsets in pediatric cholestatic liver disease at the time of liver transplant that are distinct from non-diseased M? by leveraging the ability of single cell RNA-sequencing (scRNA-seq). We have defined these M? subsets as lipid-associated M? (high expression of genes involved in lipid metabolism), monocyte-like M? (expression of genes also identified in monocytes), and adaptive M? (enrichment for genes involved in lymphocyte activation). Furthermore, monocyte-like and adaptive M? subsets positively correlate with the poor prognostic signature at diagnosis established by Luo et al suggesting these subsets may drive fibrosis and activation of CD8-positive T cells implicated in disease outcome. We thereby hypothesize that serum cytokine networks drive M? polarization towards pathogenic monocyte-like and adaptive M? subsets; increased numbers of these M? subsets by histology at diagnosis will be associated with poor outcome. To investigative this hypothesis we will: 1) determine if the numbers of lipid-associated, monocyte-like, and adaptive M? at diagnosis in human BA correlate with outcome as defined by clearance of jaundice by 3 months post-Kasai Portoenterostomy (KPE), and 2) define M?-specific cytokine networks in serum that correlate with prognosis and histologic findings. This innovative research plan applies findings from previous scRNA-seq analysis to human samples available from ChiLDReN to identify M?-subset specific histologic and serum biomarkers for prognosis at the time of diagnosis. Findings from the study will advance the care of patients with BA and allow for improved prognostic counseling at the time of KPE.